A platelet's role in hemostasis is to respond to lesions in the vasculature. Dysfunction leads to bleeding diatheses or to the pathogenic thrombosis that causes strokes, heart attacks and other occlusive events which are major health issues in today's society. To understand hemostasis, it is critical to understand platelet activation. This proposal focuses on a recently defined post-translational modification of platelet proteins that seems integral to activation. O-GlcNAcylation is not a standard form of glycosylation. It is the addition of a single N-acetylglucosamine to SER or THR residues. O-GlcNAc residues appear to be reciprocal with phosphorylation on a number of proteins and thus have been tied to key intracellular signaling events in other cell types. In platelets, O-GlcNAc is present on as many as 100 proteins and its dynamic turnover is required for platelet function, specifically granule release. When cyclical exchange of O-GlcNAc is blocked by a specific inhibitor that prevents its removal, platelets fail to respond to thrombin, collagen, or A23187. The proposed experiments will expand these observations to determine which platelet proteins are modified and thus shed light on how O-GlcNAcylation affects platelet physiology. These studies will provide the initial data to directly address our long term hypothesis that dynamic changes in O-GlcNAcylation of specific platelet proteins are critical for platelet function. One Specific Aim is proposed: To identify platelet proteins which are modified with O-GlcNAc and to determine their site(s) of modification. This Aim will use O-GlcNAc-specific affinity chromatography techniques and mass spectroscopy-based proteomic analysis to identify O-GlcNAcylated proteins in both resting and stimulated platelets. Once completed/the experiments of this two year, R21 application will provide the database needed to more fully understand the role of O- GlcNAcylation in platelet function. This information will further elucidate the events essential to the regulation of hemostasis and perhaps lead to a better understanding of how platelet function can be affected by metabolic syndromes such as diabetes. [unreadable] [unreadable] [unreadable]